Resolution Two-dimensional Polyacrylamide Gel Electrophoresis Research Articles

Differential Expression of Middle Silk Gland Proteins Caused by Cold Stress in Philosamia ricini

This study reports differentially expressed proteins in the middle silkglands of normal and cold stress treated 5th instar Philosamia ricini larvae by using high resolution two-dimensional polyacrylamide gel electrophoresis. About 300 protein spots were resolved in both type of the larvae. Most of the proteins were shown to be distributed in the area from 20 to 70 kDa ranging between pH 4 and 8. Six proteins showed considerably decreased expression in cold stress treated samples. Three proteins displayed increased level of expression after cold stress treatment, while two other proteins were expressed exclusively in cold stress treated silkgland tissues.

An improved plant leaf protein extraction method for high resolution two-dimensional polyacrylamide gel electrophoresis and comparative proteomics

We report here a simple and universally applicable protocol for extracting high quality proteins from plant leaf tissues. The protocol provides improved resolution and reproducibility of two-dimensional polyacrylamide gel electrophoresis (2-DE) and reduces the time required to analyze samples. Partitioning rubisco by polyethylene glycol (PEG) fractionation provides clearer detection of low-abundance proteins. Co-extraction of interfering substances increases the sample conductivity, which results in poor electrophoretic separation. Re-extraction of PEG-fractionated samples with phenol effectively eliminated interfering substances, which results in optimal conductivity during separation in the first dimension of the isoelectric focusing. Smooth focusing reduces analysis time and provides superior resolution in 2-DE gels. Incubating the samples at −80° C instead of −20° C reduced protein precipitation time to 2–3 h. Removal of nonprotein contaminants and the use of sonication increased protein solubility without additional reagents. These changes enabled loading and separation of maximum amounts of proteins, which permitted improved protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). An immunological approach revealed that little or no ribulose-1, 5-bisphosphte bisphosphate carboxylase oxygenase was present in the PEG supernatant. In addition, low-abundance proteins, such as myelocytomatosis transcription factor (MYC) and alpha subunit of heterotrimeric guanine nucleotide-binding protein complex (Gα), were detected only in the modified PEG supernatant and not in the total protein. These results suggest that our protocol produced high quality proteins and made many low-abundant proteins available for proteomic analysis. The successful application of this protocol for analyzing the leaf proteomes of soybean, Miscanthus sinensis, barley, Chinese cabbage, peanut and tea (Camellia sinensis) suggests that it could be used for comparative proteomic analysis of a wide range of plant leaves.

Differential proteomic analysis of developmental stages of Acca sellowiana somatic embryos

Feijoa (Acca sellowiana, Myrtaceae), a native fruit species from southern Brazil and northern Uruguay, is considered to constitute a reference system for somatic embryogenesis in woody dicots. This in vitro regenerative pathway is an efficient micropropagation method, and a suitable model system for studies in plant developmental physiology. This study attempts to detect and identify proteins that are expressed during the different developmental stages of somatic embryos of A. sellowiana. Using high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE), a high degree of similarity between protein profiles of the assayed somatic embryos was observed. Of the 74 different protein spots extracted for analysis, 60 were identified by means of 2-DE/MALDI-TOF/MS. Twelve proteins were expressed in all the assayed stages. Ten proteins were expressed in the initial stages and 22 proteins were expressed in the mature developmental stages of somatic embryos. Only one protein was expressed exclusively in the torpedo stage, whereas four were expressed in the pre-cotyledonary, and none in the cotyledonary stage. The proteins identified were involved in the synthesis of phenylalanine ammonia-lyase, a conspicuous polyphenol present in the induction of feijoa embryogenic cultures. The presence of essential proteins of nitrogen metabolism, such as the cytosolic glutamine synthetase protein, was also observed. The physiological implications of these findings are discussed.

Monitoring of stress responses

New developments in the RNA analysis techniques now enable a comprehensive view on the bacterial physiology under bioprocess conditions. The DNA-chip technology allows a genome wide transcriptional profiling of bacterial cells, whose genome sequence is available. Although the analyses of microbial bioprocesses have still been somewhat limited to date, this technique has already been successfully applied in different laboratories for the investigation of stress responses of selected industrially relevant bacterial hosts. Transcriptome analyses in combination with high resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry have been extensively applied for the description of general and specific stress and starvation responses of Escherichia coli and Bacillus subtilis. The consideration of bacterial stress and starvation responses is of crucial importance for the successful establishment of an industrial large scale bioprocess. Stress genes can be used as marker genes in order to monitor the fitness of industrial bacterial hosts during fermentation processes. This chapter gives an overview of current RNA analysis techniques. The bacterial stress and starvation responses, which are of potential importance for industrial microbial bioprocesses are summarised.

Studies on middle silkgland proteins of cocoon colour sex-limited silkworm (Bombyx mori L.) using two-dimensional polyacrylamide gel electrophoresis.

Qualitative and quantitative differences in proteins expressed in the middle silkglands of male and female silkworm larvae that differ in silk colour were investigated by high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by computer assisted image analysis. About 1000 protein spots were resolved in both the sexes and most proteins were shown to be distributed in the area from 15 kDa to 70 kDa and pH 4-8. It was found that some proteins displayed higher expression in yellow cocoon, while two proteins were only expressed in female silkworm silkgland tissue through the comparison and analysis by two-D software. These proteins especially existed in female silkworm middle silkgland tissue of yellow cocoon. Furthermore, these proteins might be involved in the expression of cocoon colour phenotype

Electrophoretic characteristics of monoclonal immunoglobulin G of different subclasses

Monoclonal IgG are commonly observed in various B cell disorders, of which multiple myeloma is the most clinically relevant. In a series of serum samples, we identified by immunofixation 73 monoclonal IgG, including 63 IgG 1, 4 IgG 2, 5 IgG 3, and 1 IgG 4. The light chains were of κ type in 45 cases, and of λ type in 28 cases. These monoclonal IgG were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) in various isoelectric focusing conditions, as well as by 3-DE (2-DE of the proteins extracted from agarose after serum protein agarose electrophoresis). After 2-DE, 38 out of 73 monoclonal γ chains (52%) were visualized using immobilized pH 3–10 gradients for isoelectric focusing. In 6 cases (8%), γ chains were only detected using alkaline immobilized pH 6–11 gradients. In 3 cases (4%), 3-DE revealed monoclonal γ chains hidden by polyclonal γ chains. Finally, in 26 cases (36%), no monoclonal γ chains were clearly visualized. Sixty-one monoclonal light chains (84%) were detected using immobilized pH 3–10 gradients, whereas 12 (16%) were not. Monoclonal γ chains and light chains were highly heterogeneous in terms of p I and M r. However, a statistically significant correlation ( P<0.05) was observed between the position of the monoclonal IgG in agarose gel and the p I of their heavy and light chains ( R=0.733, multiple linear regression). Because of the extreme diversity of their heavy and light chains, it appears that a classification of monoclonal IgG based only on their electrophoretic properties is not possible.

Identification of specific proteins in different lymphocyte populations by proteomic tools

The solubilized proteins of purified CD19(+) (B), CD8(+) (T) as well as CD4(+) (T) lymphocytes were separated by high resolution two-dimensional polyacrylamide gel electrophoresis, and the gels were analyzed using Melanie 3.0. Nine gels were studied, three for each lymphocyte population. After image analysis, 1411 +/- 73 spots (mean + SD) were detected. The protein pattern of B lymphocytes segregated from the one of T lymphocytes by ascendant heuristic clustering analysis. In addition, computer analysis separated CD8(+) from CD4(+) lymphocytes. When a search was performed in order to detect subsets of specific spots (presence vs. absence), a group of three spots, detected in the area of the protein maps corresponding to isoelectric point (pI) of 5.2 to 5.4 and molecular weight (M(r)) of 50 to 51 kDa, were found in both CD8(+) and CD4(+) cells, but not in CD19(+) cells. Mass spectrometry analysis revealed that these spots were associated with several proteins such as vimentin, tubulin, desmin and cytokeratin. Two spots, located in the area of the gel corresponding to pI of about 5.0 and a M(r) of 30 kDa, appeared as CD8(+) cell associated. Mass spectrometry analysis showed that the two spots were related to the same non-identified protein. Moreover internal peptides sequences matched with two human expressed sequence tags: gi/9759776, gi/12798420. No spots were found as only B cell associated.

Identification of specific proteins in different lymphocyte populations by proteomic tools

The solubilized proteins of purified CD19+ (B), CD8+ (T) as well as CD4+ (T) lymphocytes were separated by high resolution two-dimensional polyacrylamide gel electrophoresis, and the gels were analyzed using Melanie 3.0. Nine gels were studied, three for each lymphocyte population. After image analysis, 1411 ± 73 spots (mean + SD) were detected. The protein pattern of B lymphocytes segregated from the one of T lymphocytes by ascendant heuristic clustering analysis. In addition, computer analysis separated CD8+ from CD4+ lymphocytes. When a search was performed in order to detect subsets of specific spots (presence vs. absence), a group of three spots, detected in the area of the protein maps corresponding to isoelectric point (pI) of 5.2 to 5.4 and molecular weight (Mr) of 50 to 51 kDa, were found in both CD8+ and CD4+ cells, but not in CD19+ cells. Mass spectrometry analysis revealed that these spots were associated with several proteins such as vimentin, tubulin, desmin and cytokeratin. Two spots, located in the area of the gel corresponding to pI of about 5.0 and a Mr of 30 kDa, appeared as CD8+ cell associated. Mass spectrometry analysis showed that the two spots were related to the same non-identified protein. Moreover internal peptides sequences matched with two human expressed sequence tags: gi|9759776, gi|12798420. No spots were found as only B cell associated.

Oolemmal proteomics

Fertilization is defined as a series of gametic interactions in which capacitated sperm must first penetrate the egg vestments and then bind to and fuse with the egg plasma membrane (oolemma). The molecular basis of sperm–egg binding and fusion has yet to be elucidated due, in part, to how little is known about the array of proteins residing on the oolemma. Proteomics is an emerging area of research that directly evaluates protein expression by resolving, identifying, quantitating, and characterizing proteins utilizing a variety of techniques including high resolution two-dimensional polyacrylamide gel electrophoresis (2D PAGE), tandem mass spectrometry, and computer analysis. Our research group has utilized 2D PAGE to begin building a mouse oocyte proteomic database, with over 500 silver-stained proteins being resolved and digitized to date. Cell-surface labeling with biotin has identified a subset of 80 putative egg surface proteins. Amino acid microsequences from over 30 of the surface-labeled proteins has been obtained by tandem mass spectrometry. Sequences from eight of these proteins do not match any sequences from protein and DNA databanks, indicating that these proteins are novel. Our major current research goal is to clone, characterize, and express the novel proteins that are shown to be ovary-specific and investigate their functional roles in sperm–egg interaction.

Phosphoamino acid analysis.

Phosphorylation of amino acid residues in proteins plays a major role in biological systems. Often, phosphorylation acts as a molecular switch controlling the protein activity in different pathways as in metabolism, signal transduction, cell division etc. Therefore, identification of phosphoamino acids in proteins is an important task in protein analysis. Since the introduction of high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) a separation of phosphorylated and dephosphorylated protein species is possible. The identification of phosphorylation sites from preparative 2-D gels need very sensitive mass spectrometry methods and a specific enrichment of the phosphoprotein or -peptide.

Preliminary immunohistochemical characterization of a monoclonal antibody (pro:4-216) prepared from human prostate cancer nuclear matrix proteins

Objectives A nuclear matrix protein (PC-1) was previously identified and reported to be present only in human prostate cancer but absent in tissue from the same prostate containing either benign prostatic hyperplasia (BPH) or normal prostate tissue. The PC-1 protein was identified by high resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and exhibited a molecular mass of 56 kDa and an isoelectric point of 6.58. This work investigates the immunohistochemical characterization of PRO:4-216, a monoclonal antibody to PC-1. Methods Areas of the 2D-PAGE gels containing the human prostate cancer nuclear matrix proteins near PC-1 were isolated, eluted, and injected into mice to develop monoclonal antibodies. Antibodies were screened by immunofluorescence for nuclear reactivity to a human prostate cancer cell line (LnCaP) and by 1D and 2D Western blots for reactivity with prostate cancer nuclear matrix proteins. Monoclonal antibodies from the selected clones were affinity purified. The monoclonal antibody PRO:4-216 was used to analyze frozen tissue from 20 cancerous, 22 BPH, and 22 normal regions from fresh human prostate specimens. Tissue sections were analyzed for their immunohistochemical (IHC) (horseradish peroxidase) staining. Results Using a reference value for positive staining at an IHC score of greater than 50, 85% (17 of 20) of the cancerous, 5% (1 of 22) of the BPH, and 9% (2 of 22) of the normal prostate tissues stained positive. The one BPH and two normal tissues that stained positive were taken from prostates in which the adjacent cancerous tissue also demonstrated high IHC scores (greater than 225). Conclusions These data demonstrate nuclear reactivity on fresh frozen human prostate cancer tissue for the monoclonal antibody PRO:4-216. PRO:4-216 may aid in distinguishing normal prostate and BPH from cancerous tissue.

Cortical Brain Synaptosome Protein Map by High Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis (2-D PAGE) 1275

Cortical Brain Synaptosome Protein Map by High Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis (2-D PAGE) 1275

Analysis of protein patterns during shoot initiation in cultured Pinus radiata cotyledons

Patterns of protein synthesis were analysed during the early stages of culture of excised cotyledons of Pinus radiata grown under shoot-inductive and non-inductive conditions. The use of high resolution two-dimensional Polyacrylamide gel electrophoresis (2D-PAGE) enabled the analysis of the changes in polypeptide content of, and synthesis in, the cotyledons. A total of 54 polypeptides were identified, whose synthesis and accumulation were affected by the culture conditions. These were divided into seven classes based on their pattern of appearance in the different cultures. One class consisted of three polypeptides whose synthesis and/or accumulation was specifically enhanced in, but not unique to, shoot-forming (SF) cotyledons. The largest class consisted of 29 polypeptides whose synthesis appeared to be specifically repressed in the SF cotyledons, suggesting that part of the role of cytokinin in inducing shoot formation is to repress maturation-related changes in the SF tissues. The remaining five classes did not display a pattern of expression that could be related directly to the organogenic process.

De novo synthesis of glucocorticoid hormone regulated inner ear proteins in rats

Changes of rat inner ear de novo protein synthesis in response to dexamethasone (DEX), a synthetic glucocorticoid, have been analyzed by high resolution two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D-SDS-PAGE) and fluorography. Two proteins ( M r 41 000 and 35 000) were amplified and one protein ( M r 47 000) was suppressed by DEX in a cochlear culture medium. In the culture medium conditioned by vestibular tissue, three proteins ( M r 67 000, 57 000 and 50 000) were amplified after DEX administration. In cochlear and vestibular tissues, glucocorticoid-responsive protein synthesis was down-regulated by DEX, including two proteins ( M r 39 000 and 35 000) in the cochlea and five proteins ( M r 80 000, 64 000, 59 000, 56 000 and 40 000) in the vestibule. The regulation of these inner ear proteins by DEX suggests that glucocorticoid may play an important role in normal inner ear microhomeostasis, as well as in the treatment of some inner ear disorders.

Lung tumor cells: a multivariate approach to cell classification using two-dimensional protein pattern.

High resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a powerful research tool for the analytical separation of cellular proteins. The qualitative and quantitative pattern of polypeptides synthesized by a cell represents its phenotype and thus defines characteristics such as the morphology and the biological behavior of the cell. By analyzing and comparing the protein patterns of different cells it is possible to recognize the cell type and also to identify the most typical features of these cells. In applied pathology it is often difficult to identify the tissue of origin and the stage or grade of a neoplasia by cellular morphology analyzed by classical or immunostaining procedures. The protein pattern itself is the most characteristic feature of a cell and should therefore contribute to the identification of the cell type. For this reason we separated protein fractions originating from different lung tumor cell lines using 2-D PAGE and we compared the resulting patterns on a multivariate statistical level using correspondence analysis (CA) and ascendant hierarchical clustering (AHC). The results indicate that (i) protein patterns are highly typical for cells and that (ii) the comparison of the protein patterns of a set of interesting cell types allows the identification of potentially new marker proteins. 2-D PAGE is thus a unique and powerful tool for molecular cytology or histopathology, unveiling the protein expression level of tissues or cells.

Chamber-specific expression of human myocardial proteins detected by two-dimensional gel electrophoresis.

High resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by computer-assisted image analysis (PDQUEST) was used to screen atrial and ventricular protein patterns for quantitative and qualitative differences in protein expression. Myocardial proteins from left ventricular (LV) and right atrial (RA) samples from end-stage, failing explanted hearts and from a healthy donor heart (control) were separated by 2-D large gel electrophoresis. Ten RA versus ten LV gels from explanted dilated cardiomyopathic (DCM) hearts were analyzed for quantitative differences in their spot patterns. Of the 197 spots matched to every gel, 40 spots differed significantly in intensity between RA and LV for DCM patients. A larger number of atrial and ventricular gels (20 RA, 20 LV) from DCM patients and from a healthy donor heart (4 RA, 4 LV gels) were analyzed for qualitative differences in protein expression. Three protein spots (SSP 1120: M(r)/pI:20.5 kDa/4.6; SSP 1119: M(r)/pI:20.6 kDa/4.5; SSP 0117:M(r)/pI:20.7/ < 4.5) that are present in all RA gels for DCM patients are absent in all LV gels. Two protein spots (SSP 0112: M(r)/pI:17.2 kDa,/ < 4.4; SSP 0114:M(r)/pI:17.6 kDa/ < 4.4) occur only in all LV gels but not in the RA gels. These five qualitatively differing spots are identical in DCM patients and in the healthy donor heart. Some of the differing spots were internally sequenced and identified as myosin light chain isoforms (myosin light chain 2, atrial; myosin light chain 2, ventricular; myosin light chain 1, atrial) with the Protein Identification Resource (PIR) accession numbers A44451, S03708, A30881, respectively. Additionally, phosphoglycerate mutase (PIR: JQ0750) and ATP synthase alpha chain (PIR: S17193) were identified. Thus, quantitative and qualitative differences between atrial and ventricular protein patterns were identified by 2-D PAGE. A characteristic distribution of myosin light chains between atrial and ventricular human myocardium was found using our approach.

Regulation of phosphoprotein p18 in leukemic cells. Cell cycle regulated phosphorylation by p34cdc2 kinase.

p18 is a phosphoprotein that is expressed at very high levels in leukemic cells, at moderately high levels in proliferating normal lymphocytes, and at low levels in quiescent lymphocytes. Induction of terminal differentiation of leukemic cells in culture results in a decrease in cellular proliferation. These phenotypic changes are associated with rapid phosphorylation of p18, followed by a more gradual decrease in the level of its mRNA expression. More than 12 different phosphorylation products of p18 have been identified in different cells by high resolution two-dimensional polyacrylamide gel electrophoresis. Previous studies have suggested that p18 may be a substrate for protein kinase C in some cellular processes and protein kinase A in others. In this report, we show that the phosphorylation of p18 increases as cells progress toward the G2-M phases of the cell cycle in proliferating leukemic cells. We have examined the hypothesis that the putative role of p18 in cellular proliferation may be mediated by its involvement in the p34cdc2 kinase signal transduction pathway. We have produced recombinant p18 in bacterial cells and shown that it can be phosphorylated in vitro by purified p34cdc2 kinase with a stoichiometry of 0.86 mol of PO4/mol of substrate. We have used site-directed mutagenesis to demonstrate that the site of p34cdc2 phosphorylation is the serine at position 38. This same site has previously been shown to be phosphorylated in vivo in bovine brain along with another serine at position 25. The observation that p18 gets phosphorylated in the G2-M phases of the cell cycle and the demonstration that p18 is phosphorylated efficiently by p34cdc2 kinase in vitro at a residue that is also phosphorylated in vivo support the hypothesis that p18 may be a physiologic substrate for p34cdc2 kinase in vivo. We have also examined the effect of inhibiting the expression of p18 on cell cycle progression. These experiments demonstrated that antisense inhibition of the expression of p18 in K562 erythroleukemia cells is associated with a decrease in cellular proliferation and accumulation of cells in the G2-M phases of the cycle. The implications of these findings to the proposed role of p18 in the regulation of cellular proliferation are discussed.

Polypeptide pattern of human breast epithelial cells following human chorionic gonadotropin (hCG) treatment.

Numerous attempts have made to describe the particular protein pattern of malignant cells by using high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The placental hormone human chorionic gonadotropin (hCG) inhibits tumor initiation and progression in experimental animals and has an inhibitory effect on the proliferation of human breast epithelial cells (HBEC) in vitro. The inhibitory effect on the immortalized HBEC MCF-10F is accompanied by the immunocytochemical expression of inhibin alpha and beta subunits by treated cells. With the purpose of clarifying the molecular mechanisms involved in this effect, the pattern of protein synthesis and mRNA were studied by 2-D PAGE in the immortalized HBEC MCF-10F cells treated in vitro 1001U for 24 h. The effect of hCG treatment on the synthesis of MCF-10F cells was monitored by labeling both control and treated cells with [S35]methionine and separation by 2-D PAGE. At least 11 proteins were preferentially synthesized and five specific polypeptides were decreased in hCG treated cells in comparison with controls. The hCG induced at least four new mRNAs which encoded protein in the molecular mass range of 24-72 kDa. It also increased the expression of at least six mRNAs and reduced the expression of least four mRNAs in comparison with control cells. The hCG-treated cells actively synthesized a 33-kDa polypeptide which was not present in control cells. The nature of this hCG-inducible 33 kDa protein elucidated by immunoprecipating [S35]methionine-labeled proteins with antisera directed against rat inhibin subunit alpha and beta b.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-dimensional polyacrylamide gel electrophoresis in experimental hepatocarcinogenesis studies.

High resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in combination with computer-assisted densitometry was used to analyze sequential changes in polypeptide expression during chemically (aflatoxin Bl; AFB), spontaneously, and oncogene (v-Ha-ras, v-raf, and v-raflv-myc)-induced experimental rat hepatocarcinogenesis. Two-dimensional mapping of [35S]methionine and [32P]orthophosphate-labeled whole cell lysate and nuclear polypeptides revealed subsets of polypeptides specific for each transformation modality in the in vitro rat liver epithelial (RLE) transformation model. Many of the observed changes in whole cell lysate preparations were localized to specific subcellular organelles. Significant alterations in the expression of the extracellular matrix protein, fibronectin, as well as tropomyosin- and intermediate filament-related polypeptides (vimentin, beta-tubulin, cytokeratins 8, 14, and 18, and actin) were observed among the various transformant cell lines. Whereas alterations in the tropomyosin isoforms appeared to be transformation specific, concomitant modulation of intermediate filament expression was related more to the differentiation state of the individual cell lines than to the transformed phenotype. To integrate protein and DNA information of polypeptides believed to be critically involved during cellular transformation, N-terminal amino acid microsequencing of selected nuclear polypeptides was performed. Preliminary results suggest that N-terminal blockage of rat liver epithelial nuclear proteins to be minor (approximately 20%) with sequencing sensitivity of one pmol. These studies extend our on-going efforts toward the establishment of computerized database of rat liver epithelial cellular proteins (Wirth et al., Electrophoresis, 1991, 12, 931-954) to aid in the delineation of polypeptides critically involved in cellular growth and differentiation as well as transformation.

Non-cytopathic infection of rhabdomyosarcoma cells by coxsackie B5 virus.

Infection of rhabdomyosarcoma (RD) cells by coxsackie B5 virus (CBV5) was non-cytopathic, although low titres of infectious virus were produced after 24 h post-infection. The extent of CBV5 replication in RD cells increased after sequential passage of the virus in these cells. The RD cells from the first cycle of CBV5 infection were recovered and maintained in culture for 3 months (equivalent to 21 passages) releasing infectious virus throughout this period; these cells were considered to be persistently infected with CBV5 and were designated piRD cells. Coxsackie virus antigen was demonstrated in a small proportion of piRD cells by immunofluorescence staining. High resolution two-dimensional polyacrylamide gel electrophoresis was used to analyse the intracellular proteins prepared from piRD cells, three proteins were detected which were absent in uninfected RD cells. These new proteins were similar in charge to virus proteins induced during CBV5 lytic infection of HEp-2 cells. Quantitative densitometry of 2-dimensional protein profiles of piRD and uninfected cells showed no significant disruption of RD cell protein synthesis by the persistent virus infection. Three cloned cell lines were recovered from piRD cells, none of which showed evidence of infectious virus or virus-induced protein synthesis suggesting that the parental cell line was a carrier culture for CBV5.